The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
The accuracy of gene expression evaluation is influenced by the quantity and quality of starting RNA. Purity and integrity of RNA are critical elements for the overall success of RNA-based analyses. Starting with low quality RNA may strongly compromise the results of downstream applications which are often labour-intensive, time-consuming and highly expensive. It is therefore preferable to use high-quality intact RNA as a starting point in molecular biological as well as in diagnostic applications. The integrity of RNA should be checked in applications such as quantitative RT-PCR, RNA sequencing, micro-arrays, ribonuclease-protection-assay, in situ hybridization, northern blot analysis, RNA mapping, in vitro translation, cDNA library construction and any kind of sequencing or array applications. This issue is particularly important in clinical applications with unique or limited tissue material, (for example, tissue obtained after surgery), where a reliable RNA quantification is required.
To date, there have been methods developed which enable one to assess the quality of an RNA population of interest in order to determine whether it is of sufficient quality to use for analysis purposes. For example, to determine the purity of RNA, the OD260nm/OD280nm ratio can be taken into account, although this parameter only provides information about protein or phenol contamination, and does not give appropriate and full information about RNA integrity. For decades, the only way to determine the degradation level of RNA was the use of agarose gel-based electrophoresis, but this method is variable, inaccurate, time consuming and cost intensive.
Several methods for assessing RNA integrity are based on measuring the number of different RNA species, of the same or different lengths, or different segments of the same RNA species, and deriving a number which is related to RNA integrity. The best example is the 3′:5′ method which measures by PCR the Cq values obtained from amplification of a 3′ and a 5′ segment of an RNA molecule and uses the ratio of amplicon numbers so obtained as a measure of RNA integrity.
Automated platforms for the assessment of RNA quality are also used. Currently, two automated systems are available for this purpose: the Experion (Bio-Rad Laboratories, Hercules, Calif., USA), and the 2100 Bioanalyzer (Agilent Technologies, Palo Alto, Calif., USA). Both systems are based on an automated and miniaturized electrophoresis system, realized by Lab-on-chip technology. Both platforms determine RNA quality by using either the ribosomal 28S/18S ratio, or a numerical system which represents the integrity of RNA. Agilent Technologies offers the RIN algorithm (RNA Integrity Number) on the 2100 Bioanalyzer, and Bio-Rad recently developed a new Experion software version that offers an algorithm for calculating the RNA Quality Index (RQI). The RIN and the RQI are based on a numbering system from 1 to 10, with 1 being the most degraded RNA profile and 10 being the most intact.
However, all of the above means of assessing RNA integrity only provide a measurement on an ordinal scale: although they can rank the integrity of different samples relative to one another or to an external standard, they provide a qualitative rather than a truly quantitative measure. The number or assessment that they provide may be sufficient to indicate whether or not the integrity of RNA in a sample is sufficient to permit its further analysis, but their utility is largely limited to this purpose. What is needed is a method for measurement of RNA integrity on a ratio scale; i.e., a truly quantitative method, one which relates to the structure of RNA and enables measurements of an RNA molecule of interest to be combined with a measurement of RNA degradation to produce a quantitative measurement of the total number of the RNA molecule of interest. To date, there has been no means of achieving this.
In work leading up to the present invention, there has been developed a method for quantifying the degree of integrity of an RNA sample. More specifically, the integrity of RNA in a sample is quantified in terms of the probability that a nucleotide is damaged. This quantitative information is useful both in its own right and for use in correcting, for the degree of degradation, subsequently obtained RNA expression results.
The method of the present invention therefore has a wide range of potential applications both in terms of quantifying RNA integrity, per se, in a biological sample and, further, in terms of enabling the correction, and thereby accurate quantification, of mRNA expression levels of a specific RNA genus of interest. In terms of diagnostic and prognostic applications which rely on an analysis of changes to RNA levels, such as mRNA levels, the development of the present method now enables one to achieve a level of accuracy not previously available, and thereby overcomes currently existing diagnostic and prognostic limitations in relation to the utility of RNA data previously generated.